Use of gene editing to generate universal tcr re-directed t cells for adoptive immunotherapy

Abstract

The present invention includes compositions and methods for a modified immune cell or precursor cell thereof comprising an inducible expression system. Also provided are gene edited modified immune cells suitable for T cell therapy. Methods of treatment using modified immune cells of the present invention are also provided.

1 . A genetically modified immune cell resistant to immune-cell exhaustion comprising an exogenous nucleic acid encoding an exogenous receptor under the control of an inducible gene expression system, wherein the inducible gene expression system comprises:
 (a) a first nucleic acid comprising a constitutive promoter operably linked to a nucleic acid sequence encoding a transactivator protein;   (b) a second nucleic acid comprising an inducible promoter operably linked to a nucleic acid sequence encoding the exogenous receptor; and   (c) the first nucleic acid and the second nucleic acid are on the same expression construct;   wherein the exogenous receptor selectively binds to a tumor antigen expressed on a tumor; and   wherein when an induction agent is periodically administered to the genetically modified immune cell, the inducible gene expression system is periodically induced and the exogenous receptor is periodically expressed on the surface of the immune cell.   
     
     
         2 . The genetically modified immune cell of  claim 1 , wherein:
 (a) the inducible gene expression system comprises a bidirectional expression construct, and   (b) the second nucleic acid is in reverse orientation to the first nucleic acid.   
     
     
         3 . The genetically modified immune cell of  claim 1 , wherein the transactivator protein is selected from the group consisting of a reverse Tet repressor (rTetR), a reverse tetracycline-controlled transactivator protein (rtTA), and a Tet-On 3G transactivator protein. 
     
     
         4 - 5 . (canceled) 
     
     
         6 . The genetically modified immune cell of  claim 1 , wherein the inducible promoter comprises;
 (a) a Tet operator sequence;   (b) one or more repeats of the Tet operator sequence; or   (c) a TRE3GS promoter.   
     
     
         7 - 8 . (canceled) 
     
     
         9 . The genetically modified immune cell of  claim 1 , wherein the constitutive promoter:
 (a) drives constitutive expression of the transactivator protein; or   (b) is selected from the group consisting of a human phosphoglycerate kinase 1 (PGK1 promoter), and a human elongation factor 1 alpha (EFla) promoter.   
     
     
         10 - 12 . (canceled) 
     
     
         13 . The genetically modified immune cell of  claim 1 , wherein the induction agent is tetracycline, doxycycline, or an analog thereof. 
     
     
         14 - 17 . (canceled) 
     
     
         18 . The genetically modified immune cell of  claim 1 , wherein the exogenous receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). 
     
     
         19 . The genetically modified immune cell of  claim 18 , wherein the exogenous TCR:
 (a) is selected from the group consisting of a wild-type TCR, a high affinity TCR, and a chimeric TCR; or   (b) comprises at least one disulfide bond.   
     
     
         20 - 22 . (canceled) 
     
     
         23 . The genetically modified immune cell of  claim 18 , wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, and wherein:
 (a) the antigen-binding domain is selected from the group consisting of an antibody, an scFv, and a Fab;   (b) the transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence and transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154;   (c) the intracellular domain comprises at least one co-stimulatory domain selected from the group consisting of co-stimulatory domains of proteins in the TNFR superfamily, CD28, 4-IBB (CD137), OX40 (CD134), PD-I, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CDS, ICAM-1, LFA-1, Lek, TNFR-1, TNFR-11, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3; and/or   (d) the intracellular domain comprises an intracellular domain selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain, FcγRIII, FcsRI, a cytoplasmic tail of an Fe receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d.   
     
     
         24 - 29 . (canceled) 
     
     
         30 . The genetically modified immune cell of  claim 1 , wherein the genetically modified immune cell is an allogeneic or an autologous human T cell. 
     
     
         31 - 33 . (canceled) 
     
     
         34 . The immune cell of  claim 1 , wherein the modified immune cell further comprises an insertion and/or deletion in one or more gene locus each encoding an endogenous immune protein, and wherein the insertion and/or deletion is capable of downregulating expression of the endogenous immune protein. 
     
     
         35 . The immune cell of  claim 34 , wherein the insertion and/or deletion is the result of a CRISPR/Cas9 system-mediated gene editing;
 wherein the CRISPR/Cas9 system comprises a guide RNA, and   wherein the guide RNA comprises a guide sequence that is complementary with a target sequence of the endogenous immune protein.   
     
     
         36 - 37 . (canceled) 
     
     
         38 . The genetically modified immune cell of  claim 35 , wherein:
 (a) the endogenous immune protein is selected from the group consisting of TRAC, TRBC, B2M, CIITA, and PD1,   (b) the target sequence is within the TRAC gene and wherein the guide RNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 85-97,   (c) the target sequence is within the TRBC gene and wherein the guide RNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 1-24,   (d) the target sequence is within the B2M gene and wherein the guide RNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 73-84,   (e) the target sequence is within the CIITA gene and wherein the guide RNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 25-48, and/or   (f) the target sequence is within the PD1 gene and wherein the guide RNA comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 49-72.   
     
     
         39 - 40 . (canceled) 
     
     
         41 . The genetically modified immune cell of  claim 1 , further comprising a switch receptor. 
     
     
         42 . The genetically modified immune cell of  claim 41 , wherein the switch receptor is selected from the group consisting of PD1-CTM-CD28, PD1-PTM-CD28, TGFβR-IL12Rβ1, TGFβR-IL12Rβ2, and PD1 A132L -PTM-CD28. 
     
     
         43 - 65 . (canceled) 
     
     
         66 . A method of generating a population of modified T cells resistant to immune-cell exhaustion, the method comprising:
 (a) stimulating a population of isolated T cells to generate a population of stimulated T cells;   (b) introducing into the population of stimulated T cells a nucleic acid comprising an inducible TCR or CAR expression system to generate a population of modified T cells;   wherein the inducible gene expression system comprises:
 (i) a first nucleic acid comprising a constitutive promoter operably linked to a nucleic acid sequence encoding a transactivator protein; 
 (ii) a second nucleic acid comprising an inducible promoter operably linked to a nucleic acid sequence encoding an exogenous TCR or a CAR; and 
 (iii) the first nucleic acid and the second nucleic acid are on the same expression construct; 
   (c) introducing into the population of modified T cells one or more polypeptides and/or nucleic acids capable of downregulating expression of an endogenous immune protein to generate a population of gene edited modified T cells;   (d) depleting CD3 +  T cells from the population of gene edited modified T cells and isolating a population of CD3− gene edited modified T cells; and   (e) contacting the population of CD3− gene edited modified T cells periodically with an induction agent to periodically induce expression of the exogenous TCR or CAR, thereby generating a population of modified T cells that are resistant to immune-cell exhaustion.   
     
     
         67 . The method of  claim 66 , wherein:
 (a) stimulating a population of isolated T cells comprises contacting the population of isolated T cells with an anti-CD3 antibody and/or an anti-CD28 antibody, and   (b) depleting CD3 +  T cells from the population of gene edited modified T cells comprises removing the anti-CD3 antibody and/or the anti-CD28 antibody.   
     
     
         68 - 70 . (canceled) 
     
     
         71 . The method of  claim 66 , further comprising introducing into the population of stimulated T cells a nucleic acid encoding a switch receptor. 
     
     
         72 . The method of  claim 66 , wherein the method comprises:
 (a) stimulating the population of T cells with CD3 and/or CD28,   (b) transducing the population of T cells with a Tet-On gene inducible gene expression system for the exogenous TCR or CAR expression, wherein the Tet-On inducible gene expression system comprises a reverse Tet transactivator (rtTA) fusion protein and at least one promoter fused downstream of at least one Tet-operator sequence,   (c) electroporating the population of T cells with a CRISPR/Cas9 and a guide RNA to downregulate the expression of the endogenous immune protein, wherein the guide RNA comprises a guide sequence that is complementary with a target sequence of the endogenous immune protein,   (d) depleting the CD3+ T cells,   (e) harvesting the CD3− T cells, and   (f) contacting CD3− T cells periodically with doxycycline to periodically induce the exogenous TCR or CAR expression.   
     
     
         73 - 76 . (canceled) 
     
     
         77 . A method of preventing T cell exhaustion in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising the genetically modified immune cell of  claim 1  and a pharmaceutically acceptable carrier; and an inducing agent, wherein the inducing agent induces expression of the exogenous receptor at a tumor site in the subject. 
     
     
         78 . The method of  claim 77 , wherein:
 (a) the immune cell is contacted with the inducing agent prior to administration of the immune cell to the subject; or   (b) the inducing agent is tetracycline, doxycycline or an analog thereof.   
     
     
         79 . The method of  claim 77 , further comprising:
 (a) a step of continual administration of the induction agent to the subject to induce expression of the exogenous receptor at a tumor site within the subject;   a step of withholding administration of the induction agent to the subject to reduce expression of the exogenous receptor within the subject, thereby preventing T cell exhaustion; or   (c) a step of re-administering the induction agent to the subject to re-induce expression of the exogenous receptor within the subject.   
     
     
         80 - 82 . (canceled) 
     
     
         83 . The method of  claim 71 , wherein the switch receptor is selected from the group consisting of PD1-CTM-CD28, PD1-PTM-CD28, PD1 A132L -PTM-CD28, TGFβR-IL12Rβ1 and TGFβR-IL12Rβ2.